Characterization of Fluorescein-Based Monoboronate Probe and Its Application to the Detection of Peroxynitrite in Endothelial Cells Treated with Doxorubicin

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• 作者：Karolina Dębowska ; Dawid Dębski ; Bartosz Michałowski ; Agnieszka Dybala-Defratyka ; Tomasz Wójcik ; Radosław Michalski ; Małgorzata Jakubowska ; Anna Selmi ; Renata Smulik ; Łukasz Piotrowski ; Jan Adamus ; Andrzej Marcinek ; Stefan Chlopicki ; Adam Sikora
• 刊名：Chemical Research in Toxicology
• 出版年：2016
• 出版时间：May 16, 2016
• 年：2016
• 卷：29
• 期：5
• 页码：735-746
• 全文大小：660K
• 年卷期：0
• ISSN：1520-5010

文摘

Boronate probes have emerged recently as a versatile tool for the detection of reactive oxygen and nitrogen species. Here, we present the characterization of a fluorescein-based monoboronate probe, a 4-(pinacol boronate)benzyl derivative of fluorescein methyl ester (FBBE), that proved to be useful to detect peroxynitrite in cell culture experiments. The reactivity of FBBE toward peroxynitrite as well hypochlorite, hydrogen peroxide, and tyrosyl hydroperoxide was determined. Second-order rate constants of the reactions of FBBE with peroxynitrite, HOCl, and H₂O₂ at pH 7.4 were equal to (2.8 ± 0.2) × 10⁵ M^–1 s^–1, (8.6 ± 0.5) × 10³ M^–1 s^–1, and (0.96 ± 0.03) M^–1 s^–1, respectively. The presence of glutathione completely blocked the oxidation of the probe by HOCl and significantly inhibited its oxidation by H₂O₂ and tyrosyl hydroperoxide but not by peroxynitrite. The oxidative conversion of the probe was also studied in the systems generating singlet oxygen, superoxide radical anion, and nitric oxide in the presence and absence of glutathione. Spectroscopic characterization of FBBE and its oxidation product has been also performed. The differences in the reactivity pattern were supported by DFT quantum mechanical calculations. Finally, the FBBE probe was used to study the oxidative stress in endothelial cells (Ea.hy926) incubated with doxorubicin, a quinone anthracycline antibiotic. In endothelial cells pretreated with doxorubicin, FBBE was oxidized, and this effect was reversed by PEG-SOD and L-NAME but not by catalase.